Calmodulin is a selective mediator of Ca(2+)-induced Ca2+ release via the ryanodine receptor-like Ca2+ channel triggered by cyclic ADP-ribose.

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. We have identified calmodulin as a functional mediator of cADPR-triggered CICR through the RyRLC in sea urchin eggs. cADPR-induced Ca2+ release consisted of two phases, an initial rapid release phase and a subsequent slower release. The second phase was selectively potentiated by calmodulin which, in turn, was activated by Ca2+ released during the initial phase. Caffeine enhanced the action of calmodulin. Calmodulin did not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling.     

T-type Ca2+ current contribution to Ca2+-induced Ca2+ release in developing myocardium

In normal adult-ventricular myocardium, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is activated via Ca2+ entry through L-type Ca2+ channels. However, embryonic-ventricular myocytes have a prominent T-type Ca2+ current (ICa,T). In this study, the contribution of ICa,T to CICR was determined in chick-ventricular development. Electrically stimulated Ca2+ transients were examined in myocytes loaded with fura-2 and Ca2+ currents with perforated patch-clamp. The results show that the magnitudes of the Ca2+ transient, L-type Ca2+ current (ICa,L) and ICa,T, decline with development with the majority of the decline of transients and ICa,L occurring between embryonic day (ED) 5 and 11. Compared to controls, the magnitude of the Ca2+ transient in the presence of nifedipine was reduced by 41% at ED5, 77% at ED11, and 78% at ED15. These results demonstrated that the overall contribution of ICa,T to the transient was greatest at ED5, while ICa,L was predominate at ED11 and 15. This indicated a decline in the contribution of ICa,T to the Ca2+ transient with development. Nifedipine plus caffeine was added to deplete the SR of Ca2+ and eliminate the occurrence of CICR due to ICa,T. Under these conditions, the transients were further reduced at all three developmental ages, which indicated that a portion of the Ca2+ transients present after just nifedipine addition was due to CICR stimulated by ICa,T. These results indicate that Ca2+ entry via T-type channels plays a significant role in excitation-contraction coupling in the developing heart that includes stimulation of CICR.     

Luminal Ca2+ controls termination and refractory behavior of Ca2+-induced Ca2+ release in cardiac myocytes

Despite extensive research, the mechanisms responsible for the graded nature and early termination of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac muscle remain poorly understood. Suggested mechanisms include cytosolic Ca2+-dependent inactivation/adaptation and luminal Ca2+-dependent deactivation of the SR Ca2+ release channels/ryanodine receptors (RyRs). To explore the importance of cytosolic versus luminal Ca2+ regulatory mechanisms in controlling CICR, we assessed the impact of intra-SR Ca2+ buffering on global and local Ca2+ release properties of patch-clamped or permeabilized rat ventricular myocytes. Exogenous, low-affinity Ca2+ buffers (5 to 20 mmol/L ADA, citrate or maleate) were introduced into the SR by exposing the cells to "internal" solutions containing the buffers. Enhanced Ca2+ buffering in the SR was confirmed by an increase in the total SR Ca2+ content, as revealed by application of caffeine. At the whole-cell level, intra-SR [Ca2+] buffering dramatically increased the magnitude of Ca2+ transients induced by I(Ca) and deranged the smoothly graded I(Ca)-SR Ca2+ release relationship. The amplitude and time-to-peak of local Ca2+ release events, Ca2+ sparks, as well as the duration of local Ca2+ release fluxes underlying sparks were increased up to 2- to 3-fold. The exogenous Ca2+ buffers in the SR also reduced the frequency of repetitive activity observed at individual release sites in the presence of the RyR activator Imperatoxin A. We conclude that regulation of RyR openings by local intra-SR [Ca2+] is responsible for termination of CICR and for the subsequent restitution behavior of Ca2+ release sites in cardiac muscle.     

Sarcoplasmic reticulum Ca2+ refilling controls recovery from Ca2+-induced Ca2+ release refractoriness in heart muscle

In cardiac muscle Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca2+ influx via L-type Ca2+ channels. At present, the mechanisms underlying termination of SR Ca2+ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca2+ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca2+ release, we analyzed Na+-Ca2+ exchange currents reflecting cytosolic Ca2+ signals induced by UV-laser flash-photolysis of caged Ca2+. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, beta-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by approximately 2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 micromol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca2+ refilling mediated by the SR Ca2+-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca2+ sensitivity of CICR appears to be regulated by SR Ca2+ content, possibly resulting from a change in the steady-state Ca2+ sensitivity and in the gating kinetics of the SR Ca2+ release channels (ryanodine receptors). During Ca2+ release, the concomitant reduction in Ca2+ sensitivity of the ryanodine receptors might also underlie Ca2+ spark termination by deactivation.     

Ca2+ channel inactivation in small mesenteric arteries of WKY and SHR

BACKGROUND: This study was designed to test the hypothesis that differences exist in the inactivation properties of voltage-gated Ca(2+) channels (Ca(V)) in hypertensive arterial smooth muscle cells (ASMCs), and that these differences contribute to enhanced Ca(V) activity. METHODS: The properties of Ca(V) were studied in freshly isolated myocytes from small mesenteric arteries (SMAs) of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs) using whole-cell patch-clamp methods. RESULTS: Peak currents (I(Ca)) were larger in SHR with either 2 mmol/l Ca(2+) or Ba(2+) as the charge carrier. In WKY and SHR, the peak current was larger with Ba(2+) than with Ca(2+) with no difference in their ratio. The voltage dependence of Ca(V) activation was shifted to the left in SHR as compared to WKY for Ca(2+) but not for Ba(2+), while availability was not different. The time course of inactivation of current could be represented by two time constants, both of which were larger in SHR than in WKY and also larger for Ba(2+) than for Ca(2+), with a greater fraction of inactivation being associated with the process slower in SHR and with Ba(2+). The time courses of availability, inactivation, and recovery from inactivation were faster in SHR than in WKY in the case of Ca(2+), but there was no difference in the case of Ba(2+). CONCLUSIONS: These results demonstrate that there are differences between WKY and SHR in the inactivation properties of SMA Ca(V), and that these differences could contribute to larger steady-state currents. The differences cannot be explained merely by the presence of a larger number of identical Ca(V) complexes, and it appears likely that differences in intrinsic compositions, primary structures, and/or regulation are involved.     

Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during beta-adrenergic stimulation

We aimed to define the relative contribution of both PKA and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during beta-adrenergic receptor (betaAR) stimulation. Rat hearts were perfused with increasing concentrations of the beta-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca(2+) influx to the cell by low [Ca](o) plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [(3)H]-ryanodine binding and fast Ca(2+) release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca(2+). Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under betaAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the beta-induced increase in the channel activity as indicated by the enhancement of the [(3)H]-ryanodine binding and the velocity of fast SR Ca(2+) release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca(2+) release during betaAR stimulation.     

Prostaglandin E2-induced luteinizing hormone-releasing hormone release involves mobilization of intracellular Ca+2

The present in vitro experiments were performed to examine the involvement of Ca+2 in the mechanism by which prostaglandin E2 (PGE2) induces LHRH release from the median eminence (ME) of the hypothalamus. Stepwise decreases in the Ca+2 concentration of the incubation medium reduced the LHRH response to PGE2. Nevertheless, neither complete omission of Ca+2 (residual Ca+2 concentration, 3.5 microM) nor chelation of residual Ca+2 with EGTA prevented the stimulatory effect of the PG, suggesting that a significant portion of the PGE2 effect on LHRH release is independent of extracellular Ca+2. Blockade of Ca+2 influx with verapamil, a Ca+2 entry blocker, demonstrated that this component of the PGE2 effect is completely independent of inward Ca+2 movement. Depletion of intraterminal Ca+2 stores by exposure of the MEs to the Ca+2 ionophore A23187 in medium without Ca+2 containing EGTA almost completely obliterated the subsequent LHRH response to PGE2, indicating that normal intraterminal Ca+2 levels are important for the PGE2 effect to occur. Preloading the ME terminals with 45Ca+2 and subsequent stimulation with PGE2 demonstrated that even in the absence of extracellular Ca+2, PGE2 stimulates Ca+2 efflux from the terminals, and this Ca+2 movement occurs temporarily associated with LHRH release. Depolarization of ME terminals with 56 mM K+ in the presence of normal Ca+2 concentration resulted in massive efflux of 45Ca+2 and a greater LHRH response than that produced by PGE2, suggesting that the effect of PGE2 is not the consequence of a nonspecific general depolarization of ME nerve terminals. Thus, although a full LHRH response to (exogenous) PGE2 necessitates normal extraterminal Ca+2 concentrations, the results indicate that translocation of Ca+2 from intracellular stores is an event involved in the mechanism by which PGE2 releases LHRH.     

Role of cell volume in K+-induced Ca2+ signaling by rat adrenal glomerulosa cells

The involvement of cell volume in the K+-evoked Ca2+ signaling was studied in cultured rat glomerulosa cells. Previously we reported that hyposmosis (250 mOsm) increased the amplitude of T-type Ca2+ current and, accordingly, enhanced the Ca2+ response of cultured rat glomerulosa cells to K+. In the present study we found that this enhancement is not influenced by the cytoskeleton-disrupting drugs cytochalasin-D (20 microM) and colchicine (100 microM). Elevation of extracellular potassium concentration ([K+]e) from 3.6 to 4.6-8.6 mM induced cell swelling, which had slower kinetics than the Ca2+ signal. Cytoplasmic Ca2+ signal measured in single glomerulosa cells in response to stimulation with 5 mm K+ for 2 min showed two phases: after a rapid rise reaching a plateau within 20-30 sec, [Ca2+]c increased further slowly by approximately one third. When 5 mM K+ was coapplied with elevation of extracellular osmolarity from 290 to 320 mOsm, the second phase was prevented. These results indicate that cell swelling evoked by physiological elevation of [K+]e may contribute to the generation of sustained Ca2+ signals by enhancing voltage-activated Ca2+ influx.     

Ca2+-induced Ca2+ release in pancreatic islet beta-cells: critical evaluation of the use of endoplasmic reticulum-targeted "cameleons"

Elevated glucose concentrations cause Ca2+ influx and the exocytotic release of insulin from pancreatic islet beta-cells. Whether increases in cytosolic free Ca2+ concentration also mobilize Ca2+ from intracellular stores (Ca2+-induced Ca2+ release) is unresolved. Endoplasmic reticulum-targeted cameleons have previously been used to explore the involvement of endoplasmic reticulum (ER) Ca2+ release in these cells, albeit with differing conclusions. Cameleons comprise two spectrally shifted green fluorescent proteins, enhanced cyan and yellow fluorescent protein, whose orientation is affected by Ca2+, changing intramolecular fluorescence resonance energy transfer. By measuring pH in the cytosol and ER lumen, we demonstrate that high K+ concentrations (>20 mm) acidify both compartments in clonal MIN6 beta-cells when external bicarbonate concentrations are low (<5 mm), interfering with measurements using Ycam-2 and Ycam-4ER. However, when intracellular pH is strongly buffered (24 mm HCO3-), glucose or cell depolarization increases ER [Ca2+] monitored with Ycam-4ER. KCl-induced increases in ER [Ca2+] were diminished when intracellular stores were sensitized with 1 mm caffeine and inhibited by pretreatment with ryanodine. Furthermore, preincubation with ryanodine tended to slow the falling phase of the ER Ca2+ transient after cell depolarization with KCl and reduced the peak cytosolic [Ca2+]. By contrast, stimulation with glucose increased ER [Ca2+] both in the absence and presence of caffeine or ryanodine. These observations suggest that Ca2+-induced ER Ca2+ release can occur in beta-cells under some conditions but may not be essential for glucose-stimulated insulin secretion.     

Formation and actions of cyclic ADP-ribose in renal microvessels

Recent studies indicated that cyclic ADP-ribose (cADPR) serves as a second messenger for intracellular Ca(2+) mobilization in a variety of mammalian cells. However, the metabolism and actions of cADPR in the renal vasculature are poorly understood. In the present study, we characterized the enzymatic pathway of the production and metabolism of cADPR along the renal vascular tree and determined the role of cADPR in the control of intracellular [Ca(2+)] and vascular tone. The high performance liquid chromatographic analyses showed that cADPR was produced and hydrolyzed along the renal vasculature. The maximal conversion rate of nicotinamide guanine dinucleotide (NGD) into cyclic GDP-ribose (that represents ADP-ribosyl cyclase activity for cADPR formation) was 8.69 +/- 2.39 nmol/min/mg protein in bulk-dissected intrarenal preglomerular vessels (n = 7) and 4.35 +/- 0.13, 2.23 +/- 0.27, 2.40 +/- 0.19, and 0.31 +/- 0.02 nmol/min/mg protein, respectively, in microdissected arcuate arteries (n = 6), interlobular arteries (n = 6), afferent arterioles (n = 7), and vasa recta (n = 10). The activity of cADPR hydrolase was also detected in the renal vasculature. Using the fluorescence microscopic spectrometry, cADPR was found to produce a large rapid Ca(2+) release from beta-escin-permeabilized renal arterial smooth muscle cells (SMCs). In isolated, perfused, and pressurized small renal arteries, cADPR produced a concentration-dependent vasoconstriction when added into the bath solution. The vasoconstrictor effect of cADPR was completely blocked by tetracaine, a Ca(2+)-induced Ca(2+) release (CICR) inhibitor. These results suggest that an enzymatic pathway for cADPR production and metabolism is present along the renal vasculature and that cADPR may importantly contribute to the control of renal vascular tone through CICR.     

Leukotriene D4-induced vasoconstriction of coronary arteries in anaesthetized dogs

We examined in pentobarbital-anaesthetized dogs, the effectsof intracoronary leukotriene D₄ (LTD₄) on large vessel (circumflexartery) flow and diameter and on calculations of late diastolicand total coronary resistance. Heart rate, systolic and end-diastolicventricular pressures and the dP/dt were the haemo-dynamic variablesstudied. The peripheral ECG was obtained in lead II. LTD₄ (0.l-l0/µgkg_–1) reduced coronary diameter up to 12±3% (mean±s.e.m.).Coronary flow decreased in dose-dependent fashion up to 100%.Blood flow returned to control values within 3-15 min of LTD₄administration. Blood pressure, heart rate and ventricular pressuredid not change while LV dP/dt_max fell and filling pressure increased.The sum of ST segments and R wave voltages of the ECG increasedindicating transient myocardial ischaemia during LTD₄ inducedcoronary blood flow cessation. Small vessel and total coronaryresistance rose in a dose-dependent manner between 33-232% and50-500%, respectively. Inhibition of cyclo-oxygenase enzyme(indomethacin, 5 mg kg^–1 i.v.) and lipoxygenase enzymes(nafazatrom, 10 mg kg^–1, i.d.J had no effect on LTD₄-causedalterations in coronary flow, resistance and arterial diameter.Thus, in canine experiments the intracoronary administrationof LTD₄ can constrict coronary arteries presumably large conductivevessels. This is independent of the cyclooxygenase and additionallipoxygenase metabolites of the arachidonic acid pathway otherthan LTD₄. Therefore, the agent may contribute to cardiac dysfunctionin coronary artery disease and spasm.     

Epinephrine enhances Ca2+ current-regulated Ca2+ release and Ca2+ reuptake in rat ventricular myocytes.

The voltage dependence of the intracellular Ca2+ transients was measured in single rat ventricular myocytes with the fluorescent Ca2+ indicator dye fura-2. The whole-cell voltage clamp technique was used to measure the membrane current, and 0.9 mM fura-2 was loaded into the cell by including it in the dialyzing solution of the patch electrode. A mechanical light chopper operating at 1200 Hz was used to obtain simultaneous measurements of the intracellular Ca2+ activity with fluorescence excitation on either side of the isosbestic point (330 nm and 410 nm). The symmetry of the two optical Ca2+ signals was used as a criterion to guard against artifacts resulting, for instance, from motion. The voltage dependence of peak Ca2+ current and the Ca2+ transient measured 25 ms after depolarizing clamps from a holding potential of -40 mV were bell-shaped and virtually identical. The Ca2+ entry estimated from the integral of the Ca2+ current (0 mV, 25 ms) corresponds to a 5-10 microM increase in the total intracellular Ca2+ concentration, whereas the optical signal indicated a 100 microM increase in total intracellular Ca2+. Repolarization of clamp pulses from highly positive potentials were accompanied by a second Ca2+ transient, the magnitude of which, when summed with that measured during depolarization, was nearly constant. Ryanodine (10 microM) had little or no effect on the peak Ca2+ current but reduced the magnitude of the early Ca2+ transients by 70-90%. Epinephrine (1 microM) increased the Ca2+ current and the Ca2+ transients, accelerated the rate of decline of the Ca2+ transients at potentials between -30 and +70 mV, and reduced the intracellular [Ca2+] below baseline at potentials positive to +80 or negative to -40 mV, where clamp pulses did not elicit any Ca2+ release. Elevation of intracellular cAMP mimicked the relaxant effect of epinephrine at depolarizing potentials, whereas elevation of extracellular [Ca2+] did not. These results suggest that most of the activator Ca2+ in rat ventricular cells is released from the sarcoplasmic reticulum as a graded response to sarcolemmal Ca2+ influx. Consistent with a graded Ca2+-induced Ca2+ release we find that epinephrine increases the internal Ca2+ release by increasing the Ca2+ current. Epinephrine may also increase the Ca2+ content of the sarcoplasmic reticulum that may, in turn, increase the Ca2+-induced Ca2+ release. The relaxant effect of epinephrine appears to be caused by enhanced rate of Ca2+ resequestration and is mediated by adenylate cyclase system.     

Ca2+ -induced cell injury of cardiomyocyte

Pathogenesis of ischemia/reperfusion injury involves Ca(2+) -induced cell injury. Elevated intracellular Ca(2+) concentration at the reperfusion activates the Ca(2+) dependent protease, calpain and increases the generation of reactive oxygen species (ROS) in mitochondria, which cause cell injury in ischemia/reperfusion.     

Ca2+-induced inhibition of the cardiac Ca2+ channel depends on calmodulin

Ca2+-induced inhibition of alpha1C voltage-gated Ca2+ channels is a physiologically important regulatory mechanism that shortens the mean open time of these otherwise long-lasting high-voltage-activated channels. The mechanism of action of Ca2+ has been a matter of some controversy, as previous studies have proposed the involvement of a putative Ca2+-binding EF hand in the C terminus of alpha1C and/or a sequence downstream from this EF-hand motif containing a putative calmodulin (CaM)-binding IQ motif. Previously, using site directed mutagenesis, we have shown that disruption of the EF-hand motif does not remove Ca2+ inhibition. We now show that the IQ motif binds CaM and that disruption of this binding activity prevents Ca2+ inhibition. We propose that Ca2+ entering through the voltage-gated pore binds to CaM and that the Ca/CaM complex is the mediator of Ca2+ inhibition.     

Role of Ca2+ stores in dopamine- and PACAP-evoked growth hormone release in goldfish

Growth hormone (GH) secretion, evoked by either pituitary adenylate cyclase-activating polypeptide (PACAP) or dopamine (DA), is dependent on both voltage-sensitive calcium channels (VSCC) and cAMP signaling in goldfish. We further characterized the involvement of Ca2+ in evoked release by PACAP and DA, by examining the sensitivity of evoked GH release to perturbations of Ca2+ signaling. Both VSCC and calmodulin/calmodulin-dependent kinase are involved in PACAP signaling as had been shown for DA. In spite of this apparent dependence on VSCC, blockade of TMB-8 but not ryanodine-sensitive intracellular Ca2+ stores inhibited both PACAP- and DA-evoked GH release. Using sarcoplasmic/endoplasmic reticulum Ca-ATPases (SERCA) inhibitors, we found BHQ blocked, whereas thapsigargin (Tg) enhanced stimulated GH release, suggesting that Tg-sensitive SERCA may counteract these cAMP-mobilizing neuroendocrine regulators by sequestering [Ca2+]i. As GH secretion stimulated by two endogenous gonadotropin-releasing hormones is not affected by Tg, it appears that distinct multiple Ca2+ stores mediate the hormone releasing response to different neuroendocrine regulators.     

Evoked and spontaneous purinergic junctional Ca2+ transients (jCaTs) in rat small arteries

Confocal microscopy of fluo-4 fluorescence in pressurized rat mesenteric small arteries subjected to low-frequency electrical field stimulation revealed Ca2+ transients in perivascular nerves and novel, spatially localized Ca2+ transients in adjacent smooth muscle cells. These muscle Ca2+ transients occur with a very brief latency to the stimulus pulse (most <3 ms). They are wider (approximately 5 micro m) and last longer (t(1/2), 145 ms) than Ca2+ sparks. They are abolished by the purinergic receptor (P2X) antagonist suramin, but they are totally unaffected by the alpha1 adrenoceptor antagonist prazosin or by capsaicin (which inhibits the function of perivascular sensory nerves). We conclude that these novel Ca2+ transients represent Ca2+ entering smooth muscle cells through P2X receptors activated by ATP released from sympathetic nerves, and we therefore call them "junctional Ca2+ transients" or jCaTs. As expected from spontaneous neurotransmitter release, jCaTs also occur spontaneously, with characteristics identical to evoked jCaTs. Visualization of sympathetic neurotransmission shows that purinergic components dominate at low frequencies of sympathetic nerve fiber activation.